The rapid development of molecularly targeted probes for use in vivo has led to growing interest in clinical applications that incorporate information from these probes. In particular, there is a need for techniques to visualize these targeted probes during surgery; the probes could be used, for example, to identify tissue either for removal or preservation. This project focuses on the development of instrumentation for real-time visualization of fluorescent probes. When looking at fluorescent probes in tissue, the major difficulty is usually separating the probe fluorescence from the tissue autofluorescence. Because the autofluorescence has different spectral properties than the fluorophore, it can easily be separated using multispectral imaging, in which several full emission spectra can be taken for each pixel. Although the implementation of acousto-optic tunable filters has greatly increased the speed of these techniques, the minimum time required to acquire an image cube is still several seconds with commercially available systems. This data acquisition rate makes the use of multispectral imaging to provide real-time feedback during a surgical procedure impractical.[unreadable] [unreadable] As an alternative to multispectral imaging, aggressive filtering of both emission and excitation light can help to minimize the effects of tissue autofluorescence. Although the signal level drops as the spectral window is narrowed, this can be overcome by using a sensitive camera, such as a cooled CCD, ICCD, or even an EMCCD. Because a single spectral window is used, the data-taking rate is much faster, and could easily approach the video rate, thirty frames per second. Depending on the concentration, extinction coefficient, and quantum yield of the fluorophore, it may also be necessary to acquire simultaneous fluorescence images at two wavelengths in order to provide a correction for the autofluorescence. This simple autofluorescence correction could also be implemented with a single camera using two emission filters, but the image acquisition rate would be considerably slower.[unreadable] [unreadable] The immediate application for this instrument is the identification of peritoneal metastases in ovarian cancer, using a GSA-Rhodamine Green probe developed in NCI. We had previously assembled a simple instrument comprised of a fiber optic ring light with a narrow bandpass filter between two fiber bundles, a narrow bandpass emission filter, and a single cooled monochrome CCD camera. The sensitivity was sufficient to identify labeled metastases at an image acquisition rate of five frames per second with specificity comparable to that achieved with a multispectral system. This instrument was successfully used for mock cytoreductive surgery in a murine model of ovarian cancer, and the results were published this year. [unreadable] [unreadable] In addition, a further set of experiments were performed, using an improved experimental setup which permitted image acquisition at fifteen frames per second with enhanced sensitivity, effectively eliminating any lag in the video monitoring during surgery. Cytoreductive surgery was performed on anesthetized animals, with similar success to the previous mock surgery. In addition, we started development on a separate multispectral system to provide a white light image stack for physical registration of the multispectral image cube. The hardware developed for this application could be easily adapted to incorporate a second camera into the instrument for fluorescence guided surgery; this second camera could be used either for a second fluorescence image, in order to provide a simple autofluorescence correction, or for a pseudo-white light image of the surgical field.